The invention relates to formulations of paclitaxel and paclitaxel analogs, or conjugates thereof, as well as other hydrophobic agents.
Due to insolubility in aqueous solution, hydrophobic agents, such as paclitaxel and paclitaxel analogs, typically are either solubilized in non-aqueous or surfactant buffers or attached to hydrophilic moieties for increased solubility in aqueous solution prior to administration to a patient. Paclitaxel is commercially supplied in a formulation where each ml contains 6 mg paclitaxel, 527 mg of purified CREMOPHOR® EL (polyoxyethylated castor oil) and 49.7% (v/v) dehydrated alcohol, USP and ethanol. Prior to administration, the formulated paclitaxel is diluted in a sodium chloride/dextrose or dextrose in Ringer's solution. Because CREMOPHOR® can cause hypersensitivity (e.g., anaphylactic) reactions, patients receiving paclitaxel are premedicated with dexamethasone to reduce the occurrence of these reactions. Because of these reactions, paclitaxel is administered over 4 hours to minimize the hypersensitivity effects.
Because of the high rate of side effects due to the inclusion of CREMOPHOR® in the standard paclitaxel formulations, alternate formulations have been created. These formulations rely upon association of paclitaxel with a soluble compound. Abraxane is paclitaxel formulation where paclitaxel is bound to albumin. Liposomal paclitaxel formulations have also been proposed.
Because the existing formulations of hydrophobic agents, such as paclitaxel, either contain undesirable excipients or can be difficult to manufacture, there is a need for new formulations of such agents.
In first aspect, the invention features a composition including (a) a hydrophobic agent, paclitaxel, a paclitaxel analog, or a conjugate (e.g., ANG1005) including (i) a polypeptide vector; and (ii) a therapeutic agent selected from the group consisting of paclitaxel and a paclitaxel analog, where the therapeutic agent is conjugated to a polypeptide, or any hydrophobic agent described herein); (b) an optional tonicity agent (e.g., sodium chloride or any tonicity agent described herein); (c) a buffering agent (e.g., glycine, lactic acid, or citric acid, or any buffering agent described herein); (d) a bulking agent (e.g., mannitol, sorbitol, or any bulking agent described herein); and (e) a solubilizing agent (e.g., polyoxyethylene ester of a fatty acid such as SOLUTOL®HS 15, or any solubilizing agent described herein), for example, where the solubilizing agent is not CREMOPHOR®. The polypeptide vector may include an amino acid sequence substantially identical (e.g., at least 70%, 80%, 90%, 95%, or 100% identical) to an amino acid sequence selected from the group consisting of SEQ ID NOS:1-105 and 107-116 (e.g., AngioPep-1 (SEQ ID NO:67); AngioPep-2 (SEQ ID NO:97), or AngioPep-7 (SEQ ID NO:112)). In certain embodiments, the buffering agent maintains the solution at a pH of less than 6 (e.g., pH 4-6). In certain embodiments, the composition further includes 0.01-10% (e.g., less than 8%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.2%, or 0.1%) DMSO. In certain embodiments, the composition is substantially free from CREMOPHOR® (e.g., free of CREMOPHOR®). The composition may be dissolved in water.
In certain embodiments, the composition comprises agents in the amounts shown in any of Tables 1-4.
TABLE 1Percentage (byCompoundnon-water weight)ANG10050.1-5%  Tonicity agent0-15%Buffering agent1-10%Bulking agent0-15%SOLUTOL ® HS 1540-75% DMSO0.01-10%  
TABLE 2Percentage (byCompoundnon-water weight)ANG10051.8-2.3%Tonicity agent 9-11%Buffer4.5-6%  Bulking agent 8-10%SOLUTOL ® HS 1569-75%DMSO0.2-1%  
TABLE 3Percentage (byCompoundnon-water weight)ANG10051.8-4.0%Buffer0.1-6%  Bulking agent 2-10%SOLUTOL ® HS 1580-95%DMSO0.2-1%  
TABLE 4Percentage (byCompoundnon-water weight)ANG10052.0-3.0%Buffer0.5-6%  Bulking agent4-7%SOLUTOL ® HS 1585-95%DMSO0.2-0.6%
In these compositions, the tonicity agent, if present, may be sodium chloride, the buffering agent may be glycine, lactic acid, or citric acid, and/or the bulking agent may be mannitol. The composition may be made up of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% 1.1, 1.2, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 3.0%, 3.2%, 3.5%, 4.0%, or 5.0% ANG1005, or any range in between any of these values. The ANG1005 may be dissolved in a sufficient amount of SOLUTOL® HS 15, and/or DMSO, which may be further diluted in an aqueous solution.
The above compositions may be present in a container that may be sealed. The container may be part of a kit that further includes instructions for use (e.g., for administering the composition for treatment of any disease such as those described herein).
In another aspect, the invention features a method of administering a composition of the above aspects to patient suffering from a disease, for example, any disease described herein such as cancer (e.g., ovary, brain, lung, liver, spleen, or kidney cancer The method includes administering to the patient the composition in an amount sufficient to treat or treat prophylactically the disease. In certain embodiments, the cancer is a brain cancer selected from the group consisting of glioblastoma, astrocytoma, glioma, meduloblastoma, and oligodendroma, neuroglioma, ependymoma, and meningioma.
In another aspect, the invention features a method for preparing a pharmaceutical composition. The method includes (a) dissolving a hydrophobic agent in a first solubilizing agent (e.g., DMSO or any such agent described herein) to form a mixture; (b) adding a second solubilizing agent (e.g., a polyoxyethylene ester of a fatty acid such as SOLUTOL® HS 15, or any such agent described herein) to the mixture of step (a); (c) optionally adding water and a buffering agent to the mixture; (d) lyophilizing mixture of step (c); where the lyophilization results in a reduction of at least 5% (e.g., 10%, 20%, 30%, 50%, 75%, 90%, 95%, or 99%) of the amount of the first solubilizing agent (e.g., to a final proportion of less than 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.5%, 2%, 3%, 4%, 5%, 8% of the total weight of the lyophilized product). In certain embodiments, the lyophilizing does not substantially reduce the amount of the second solubilizing agent. In certain embodiments, the hydrophobic agent includes paclitaxel or a paclitaxel analog. The hydrophobic agent may include or may be a conjugate including (a) a polypeptide vector and (b) an agent described herein (e.g., paclitaxel and analogs thereof), where the agent is conjugated to the vector. The polypeptide vector may be substantially identical to an amino acid sequence selected from the group consisting of SEQ ID NOS:1-105 and 107-116 (e.g., AngioPep-1 (SEQ ID NO:67); AngioPep-2 (SEQ ID NO:97), or AngioPep-7 (SEQ ID NO:112)). In particular embodiments, the conjugate is ANG1005. In certain embodiments, water and a buffering agent are added in step (c) and the step (d) lyophilizing includes (i) freezing the mixture; (ii) drying the frozen product at a first temperature and pressure sufficient to remove at least a portion (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, 99.9%, or 99.99%) of the water; and (iii) drying the product at a second temperature and pressure sufficient to remove at least a portion (e.g., at least 5% (e.g., 10%, 20%, 30%, 50%, 75%, 90%, 95%, or 99%) of the first solvent. The mixture of step (b) may be filtered prior to step (c) lyophilizing or may be placed into a vial or container prior to step (c) lyophilizing. The method may further include (c) resuspending the lyophilized product.
In another aspect, the invention features a method for producing a pharmaceutical composition including the steps (a) dissolving in DMSO a conjugate including paclitaxel or paclitaxel analog conjugated to a polypeptide vector, thereby forming a mixture; (b) adding SOLUTOL® HS 15 to the mixture; (c) adding water, a buffering agent, and optionally salt or a bulking agent to the mixture; and (d) lyophilizing the mixture under conditions which remove the water and the DMSO from the mixture. The SOLUTOL® HS 15 may be mixed with water, a buffering agent, and optionally a tonicity agent or a bulking agent prior to adding to the mixture, where the water, buffering agent, and optional tonicity agent are added in a amount which maintains solubility of the conjugate in the mixture. The buffering agent may maintain the solution at a pH between 4 and 6. The DMSO may be acidified between pH 3.5 and 4.5 prior to the step (a) dissolving. In certain embodiments, the lyophilization does not substantially reduce the amount of SOLUTOL® HS 15 in the mixture. The conjugate may include any of the polypeptides (e.g., AngioPep-2) described herein. In particular embodiments, the paclitaxel-polypeptide conjugate is ANG1005.
In another aspect, the invention features a pharmaceutical composition produced by any of the methods described above.
By “buffering agent” is meant any compound or group of compounds capable of maintaining the pH (e.g., between any of pH 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, and 13.5) of a solution within a particular range upon addition of agents that can otherwise alter the pH. Exemplary buffering agents are described herein.
By “tonicity agent” is meant any agent that alters the osmolarity of an aqueous solution (e.g., any of or any range between 10, 20, 50, 75, 100, 150, 200, 250, 300, 400, 500, 750, 1000, 1500, or 2000 mM). Ionic salts, such as sodium chloride, can be used to adjust tonicity. Additional tonicity agents are described herein.
By “bulking agent” is meant a compound that alters the physical form of a chemical composition following a dehydration or lyophilization procedure. Exemplary bulking agents are described herein.
By “solubilizing agent” is meant any solvent capable of dissolving a particular compound (e.g., a hydrophobic compound such a compound or conjugate containing paclitaxel or a paclitaxel analog). Exemplary solubilizing agents suitable for hydrophobic compounds are described herein.
By “vector” is meant a compound or molecule such as a polypeptide that can be transported into a particular cell type (e.g., liver, lungs, kidney, spleen, or muscle) or across the BBB. The vector may be attached to (covalently or not) or conjugated to an agent and thereby may be able to transport the agent into a particular cell type or across the BBB. In certain embodiments, the vector may bind to receptors present on cancer cells or brain endothelial cells and thereby be transported into the cancer cell or across the BBB by transcytosis. The vector may be a molecule for which high levels of transendothelial transport may be obtained, without affecting the cell or BBB integrity. The vector may be a polypeptide or a peptidomimetic and may be naturally occurring or produced by chemical synthesis or recombinant genetic technology.
By “conjugate” is meant a vector linked to an agent. The conjugation may be chemical in nature, such as via a linker, or genetic in nature for example by recombinant genetic technology, such as in a fusion protein with for example a reporter molecule (e.g., green fluorescent protein, β-galactosidase, Histag, etc.).
By a vector or conjugate which is “efficiently transported to a particular cell type” is meant a vector or conjugate that is able to accumulate (e.g., either due to increased transport into the cell, decreased efflux from the cell, or a combination thereof) in that cell type at least 10% (e.g., 25%, 50%, 100%, 200%, 500%, 1,000%, 5,000%, or 10,000%) greater extent than either a control substance, or, in the case of a conjugate, as compared to the unconjugated agent.
By “substantially pure” or “isolated” is meant a compound (e.g., a polypeptide or conjugate) that has been separated from other chemical components. Typically, the compound is substantially pure when it is at least 30%, by weight, free from other components. In certain embodiments, the preparation is at least 50%, 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by weight, free from other components. A purified polypeptide may be obtained, for example, by expression of a recombinant polynucleotide encoding such a polypeptide or by chemically synthesizing the polypeptide. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
A pharmaceutical composition which is “substantially free” from a substance means that the amount of a substance in the composition is less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, 0.1%, 0.05%, or 0.01% of the dry weight of a composition.
By “substantially identical” is meant a polypeptide or nucleic acid exhibiting at least 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, or even 99% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 4 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, or 100) amino acids. For nucleic acids, the length of comparison sequences will generally be at least 60 nucleotides, preferably at least 90 nucleotides, and more preferably at least 120 nucleotides, or full length. It is to be understood herein that gaps may be found between the amino acids of an analogs which are identical or similar to amino acids of the original polypeptide. The gaps may include no amino acids, one or more amino acids which are not identical or similar to the original polypeptide. Biologically active analogs of the vectors (polypeptides) of the invention are encompassed herewith. Percent identity may be determined, for example, with n algorithm GAP, BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release 7.0, using default gap weights.
By “fragment” is meant a polypeptide originating from a portion of an original or parent sequence or from an analogue of said parent sequence. Fragments encompass polypeptides having truncations of one or more amino acids, wherein the truncation may originate from the amino terminus (N-terminus), carboxy terminus (C-terminus), or from the interior of the protein. A fragment may include the same sequence as the corresponding portion of the original sequence. Functional fragments of the vector (polypeptide) described herein are encompassed by the invention. Fragments may be at least 5 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 28, 30, 35, 40, 45, 50, 60, 75, 100, or 150) amino acids. Fragments of the invention may include, for example, a polypeptide of 7, 8, 9 or 10 amino acids to 18 amino acids. Fragments may contain any of the modifications described herein (e.g., acetylation, amidation, amino acid substitutions)
A “non-naturally occurring amino acid” is an amino acid that is not naturally produced or found in a mammal.
By “agent” is meant any compound, for example, an antibody, or a therapeutic agent, a marker, a tracer, or an imaging compound.
By “therapeutic agent” is meant an agent having a biological activity. In some cases, the therapeutic agent is used to treat the symptoms of a disease, a physical or mental condition, an injury, or an infection and includes anti-cancer agents, antibiotics, anti-angiogenic agents, and molecules active at the level of the central nervous system.
By “small molecule drug” is meant a drug having a molecular weight of 1000 g/mol or less (e.g., less than 800, 600, 500, 400, or 200 g/mol).
By “subject” is meant a human or non-human animal (e.g., a mammal).
By “treating” a disease, disorder, or condition in a subject is meant reducing at least one symptom of the disease, disorder, or condition by administrating a therapeutic agent to the subject.
By “treating prophylactically” a disease, disorder, or condition in a subject is meant reducing the frequency of occurrence of (e.g., preventing) a disease, disorder or condition by administering a therapeutic agent to the subject.
By “cancer” is meant any cellular proliferation whose unique trait is the loss of normal controls which can result in unregulated growth, lack of differentiation, or ability to invade tissues and metastasize. Cancer can develop in any tissue or in any organ. Cancer is intended to include, without limitation, cancer of the brain, liver, lungs, kidney, or spleen. Additional cancers are described herein.
By “administering” and “administration” is meant a mode of delivery including, without limitation, orally, intra-arterially, intra-nasally, intraperitoneally, intravenously, intramuscularly, subcutaneously, transdermally or per os. A daily dosage can be divided into one, two or more doses in a suitable form to be administered at one, two or more times throughout a time period.
By “therapeutically effective” or “effective amount” is meant an amount of a therapeutic agent sufficient to improve, decrease, prevent, delay, suppress, or arrest any symptom of the disease or condition being treated. A therapeutically effective amount of an agent need not cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, or the disease or condition symptoms are ameliorated, or the term of the disease or condition is changed or, for example, is less severe or recovery is accelerated in an individual.
If a “range” or “group of substances” is mentioned with respect to a particular characteristic (e.g., temperature, concentration, time and the like), the invention relates to and explicitly incorporates herein each and every specific member and combination of sub-ranges or sub-groups therein. Thus, for example, with respect to a length of from 9 to 18 amino acids, is to be understood as specifically incorporating herein each and every individual length, e.g., a length of 18, 17, 15, 10, 9, and any number therebetween. Therefore, unless specifically mentioned, every range mentioned herein is to be understood as being inclusive. For example, in the expression from 5 to 19 amino acids long is to be as including 5 and 19. This similarly applies with respect to other parameters such as sequences, length, concentrations, elements, and the like.
The sequences, regions, portions defined herein each include each and every individual sequence, region, and portion described thereby as well as each and every possible sub-sequence, sub-region, and sub-portion whether such sub-sequences, sub-regions, and sub-portions are defined as positively including particular possibilities, as excluding particular possibilities or a combination thereof. For example, an exclusionary definition for a region may read as follows: “provided that said polypeptide is no than 4, 5, 6, 7, 8 or 9 amino acids. A further example of a negative limitation is the following; a sequence including SEQ ID NO:X with the exclusion of a polypeptide of SEQ ID NO:Y; etc. An additional example of a negative limitation is the following; provided that said polypeptide is not (does not include or consist of) SEQ ID NO:Z.
Other features and advantages of the invention will be apparent from the following Detailed Description, the drawings, and the claims.